R-050. Amplification of a Genetic Segment Containing a Citrate Transporter Gene Underlies Evolution of Aerobic Citrate Utilization in a Long-Term Experimental Population of Escherichia coli

Z. D. Blount1, J. E. Barrick1, S. C. Sleight1,2, R. E. Lenski1;
1Michigan State Univ., East Lansing, MI, 2Univ. of Washington, Seattle, WA.

In 1988, twelve populations of E. coli B were founded from strain REL606. These have since been evolved for 45,000+ generations of daily serial transfer in DM25, a minimal medium containing 140 μM glucose and 1,700 μM citrate. However, E. coli cannot use citrate as a carbon and energy source under the oxic conditions of the experiment. As E. coli possesses a complete TCA cycle, and most strains can ferment citrate, the inability to use citrate aerobically is attributed to a lack of citrate transporter expression under oxia. After 33,000 generations, population Ara-3 experienced a massive population expansion caused by variants capable of aerobic citrate utilization (Cit+). These variants originally evolved between 31,500 and 32,000 generations. We hypothesized that the Cit+ phenotype was caused by a mutation that altered the regulation of a citrate transporter. To test this hypothesis, we compared the genome sequences of a Cit+ variant from 32,000 generations and a Cit- variant from 32,500 generations. Sequencing was done using 454 technology, with assembly on the ancestral REL606 genome. In contrast to REL606 and the Cit- variant, the Cit+ genome showed a ~60 kb duplication within which a small, 2.9 kb segment had been amplified 4- to 8-fold. The amplified segment overlapped part of the citrate fermentation operon, with its boundaries within the distal end of the citG gene and the proximal end of the rnk gene that encodes a nucleoside kinase regulator involved in aerobic metabolism. The segment contained the entire ribonuclease I gene, and the citT gene, which encodes a citrate transporter. Segment copies were tandem, in a head-to-tail orientation placing citT downstream of the rnk promoter, likely causing CitT transporter expression during aerobic growth. Similar amplifications placing citT downstream of the rnk promoter have since been identified in multiple independent Cit+ mutants, but in no Cit- clones. The strength of the Cit+ phenotype seems to vary by the distal boundary's position relative to the rnk promoter. Our results provide further evidence of the role of gene duplication in the evolution of novel, potentially cladogenic traits.